Plant Cell Physiol. 49(5): 718–729 (2008)
doi:10.1093/pcp/pcn047, available online at www.pcp.oxfordjournals.org
ß The Author 2008. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.
All rights reserved. For permissions, please email: [email protected]
An Analysis of Long-Distance Water Transport in the Soybean
Stem Using H215O
Tomoyuki Ohya 1, *, Keitaro Tanoi 2, Yousuke Hamada 2, Hirotaka Okabe 3, Hiroki Rai 2, Junko Hojo 1,
Kazutoshi Suzuki 1 and Tomoko M. Nakanishi 2
1
National Institute of Radiological Sciences, Medical Imaging, 4-9-1, Anagawa Inage-ku, Chibashi, 265-8555, Japan
Graduate School of Agricultural and Life Sciences, The University of Tokyo, Applied Biological Chemistry, 1-1-1, Yayoi Bunkyo-ku,
Tokyo, 113-8657 Japan
3
Department of Applied Quantum Physics and Nuclear Engineering, Faculty of Engineering, Kyushu University, 744, Motooka,
Fukuoka, 819-0395 Japan
2
reviewed in Van Bel 1990). Along the symplastic xylem–
phloem route, solutes escape from xylem vessels via the pits,
are transported into the ray cells, and are finally loaded into
the sieve tubes. Via the apoplastic xylem–phloem route,
solutes are transported from the xylem vessels via a cell wall
continuum towards the phloem due to a concentration
gradient. These complex, lateral transport mechanisms have
several physiological consequences. A part of the solute was
found to be sequestered in the apoplastic or symplastic stores
and was used to buffer variations between sources and sinks
in daily and seasonal changes (Thorpe et al. 2005). The route
from xylem to phloem was exploited for the transfer of
nitrogen to the fruits of soybean plants (Layzell and LaRue
1982). It may be used to remove superfluous ions from plants
(Levi 1970). Thus, the mechanism may serve for the selective
withdrawal and transport of solutes. The mechanism is able
to meet the requirements of various tissues and organs at
specific time points (Van Bel 1990).
The question remains as to how the movement of
water as a solvent is affected by lateral transport. It has
been reported that the longitudinal transport velocity of
water through phloem and xylem was much slower than
that of phosphorus and sugar (Biddulph and Cory 1957,
Gage and Aronoff 1962, Trip and Gorham 1968). Van Bel
(1974, 1976) analyzed the uptake of a solution by tissues
surrounding the xylem vessels by simultaneous perfusion
of tritiated water (THO), sugar and amino acid through
stem segments of tomato plants. The results showed that
transport through xylem vessels may be considered as a
chromatography-like process with different longitudinal
transfer velocities for each component. Water escaped more
easily from the xylem vessels, resulting in a different
longitudinal displacement of solvent compared with solutes.
Van Bel assumed that dilution of the labeled water during
its transport, which was caused by exchange with innate
non-labeled water, is driven by diffusion as based on the
Horwitz theory (Horwitz 1958).
The present study also assumes that water was
transported in a lateral direction along the two-way route
The lateral water movement in the intact stem of a
transpiring soybean plant was analyzed quantitatively by a
real-time measurement system utilizing labeled water, H215O
and gamma ray detectors. A large volume of water escaping
from xylem vessels during its transport was detected. The
escape of water was not influenced by evaporation from the
stem surface or mass flow in the sieve tubes. It was assumed
that the total amount of water transported through xylem vessels was kept almost completely constant along the internode.
As a result, most of the escaped water was found to re-enter the
xylem vessels, i.e. water exchange occurred. The analysis of
radiographs of tritiated water suggested that the self-diffusion
effect of water was strong for lateral water movement,
although another driving force besides thermal motion was
included in the process, and that the process was also affected
by the water permeability of the plasma membrane. An
analysis based on a mathematical model showed that the net
volume of water which escaped from xylem vessels was not
dependent on the transpiration rate of the plant.
Keywords: H215O — Internode — Real-time measurement
— Soybean — Water exchange — Water relations.
Abbreviations: AFS, apparent free space; MRI, magnetic
resonance imaging; PETIS, positron-emitting tracer imaging
system; RH, relative humidity; THO, tritiated water.
Introduction
With the development of the study of transport
physiology, it has become clear that mass transport in
plants is not just a simple flow through xylem vessels and
sieve tubes, but also includes xylem–phloem exchange
during transport (Van Bel 1990, Patrick et al. 2001).
For the lateral transport, a quantitative, two-way model of
xylem–phloem transport has been proposed. It supposes
symplastic and apoplastic routes operating in parallel for the
radial transport (a detailed explanation of this model was
*Corresponding author: E-mail, [email protected]; Fax, þ81-43-206-3261.
718
Lateral water movement in soybean stems
described above. However, detailed analyses of the time
dependence of water transport by intact stems of transpiring
plants have never been accomplished. This is due to the
technical difficulties associated with the use of THO as a
water tracer in studies on water transport (Cline 1953).
THO is a radioactive tracer with beta ray energy of 19 keV.
Since this energy is too weak to penetrate plant tissues,
samples have to be extracted to measure the amount of
THO (Bull et al. 1972). Other isotope tracers, such as D2O
and H218O, which have also been used to monitor water
relations, also require destruction of plant tissue prior to
measurement by mass spectrometry (DeKroon et al. 1996,
Gan et al. 2002).
For a meaningful study of water transport, it is
imperative to observe real-time water transport in intact
stems of transpiring plants. It has been reported that the
flow rate through xylem vessels depends on the solute
concentration because the pits (the contact sites between
vessels and between vessels and the surrounding parenchyma cells) control the mass flow rate, which is dependent
on the hydrogel character of the pit membrane (Zwieniecki
et al. 2001, Zwieniecki et al. 2003). It is thought that the
change in water conductivity controlled by the pit
membrane is also caused by the substrates unloaded from
phloem as well as xylem vessels (Thompson and Zwieniecki
2005). These reports suggest that longitudinal and radial
transport of solute via xylem and phloem have a large effect
on water movement in the stem. Thus, it is important to
keep the shoot in the natural state to analyze the
longitudinal and lateral water movement in the stem. For
more conclusive studies, a measuring system was developed
that allows non-invasive detection of trace amounts of
water in plants.
MRI (magnetic resonance imaging) is a newly developed method, useful for the observation of water distribution in tissues (Ishida et al. 2000). It allows non-invasive
measurements of mass flow rates in both xylem vessels and
sieve tubes (Kuchenbrod et al. 1996, Rokitta et al. 1999,
Scheenen et al. 2002). However, the dynamic interaction
between innate water and newly absorbed water during
their upward transport in living plants is unclear due to the
limited sensitivity and time resolution of MRI.
Recently, the radioisotope tracer H215O has been
introduced to meet these shortcomings. In nuclear medicine,
H215O has been used for the analysis of regional cerebral
blood flow by positron emission tomography (PET)
(Raichle et al. 1983). 15O is a positron nuclide, i.e. positrons
are emitted from bþ decay of 15O. A pair of gamma rays,
which make an angle of 1808 to each other, are emitted as
the result of pair annihilation of the positron in question.
The energy of the gamma rays is 511 keV. This allows
measurement of H215O within living tissues with a high
signal-to-noise ratio by discriminating the energy and
719
counting the annihilation gamma rays by using a pair of
gamma ray detectors combined with a coincidence circuit.
Moreover, since the half-life of 15O is extremely short
(122 s), the experiment can be repeated using the same plant
after various treatments, which eliminates sample errors
and corroborates the reproducibility of the results.
The positron-emitting tracer imaging system (PETIS)
is a tool for visualizing the two-dimensional movement of
positron nuclides in plant (Kume et al. 1997). Recently,
H215O movement recorded with PETIS has been used for
the study of water transport in tomato and rice plants (Mori
et al. 2000, Kiyomiya et al. 2001, Nakanishi et al. 2002).
They investigated the effect of light conditions and reagents
(including NaCl, ABA and methylmercury) on water
movement in plants. PETIS, however, does not allow a
quantitative analysis due to technical difficulties, such as the
correction for relative detection efficiencies within the field
of view and the positron escape from the samples (Levin
and Hoffman 1999). Thus, the problem of quantitative
analysis of long-distance transport of water was not yet
solved. We designed a measuring system that does enable
quantitative measurement. The positional information is
maintained by downscaling the size of the gamma ray
detectors. Furthermore, the effect of the positron escape
from the samples is reduced by setting the detectors
adjacent to either side of the target site. With this system,
the volume of H215O in 1 cm of an internode could be
measured quantitatively and non-invasively, i.e. it enables
us to analyze the longitudinal and lateral water movement
in the intact stem of the transpiring plants.
Here we report the results obtained with this new
design, including a mathematical model which describes
how H215O is translocated through a stem internode.
Results and Discussion
Water which escaped from xylem vessels and the distribution
of the water
In the present study, we use the term ‘H215O’ for the
newly introduced water to distinguish this from the innate
water. A precise description of the term is that 10 g of H216O
contained about 1 10–11 g of H215O.
H215O was applied to the cut end of the stem, 10 cm
below the cotyledon, of soybean plants which were about
3 weeks old (Fig. 1). The radioactivity of H215O, i.e. the
volume of H215O, in the internode initially increased linearly.
The slope of the curve then decreased gradually (Fig. 2).
The profile of the radioactivity distribution was similar to the
results of Mori et al. (2000) obtained by PETIS. The amount
of H215O taken up by the internode exceeded the aggregate
capacity of the xylem (1.9 ml) within a few minutes of
application of H215O. The capacity of the vessels was
estimated from the measurement of their transverse
720
Lateral water movement in soybean stems
sectional area by microscopy [ ¼ 1.9 0.30 10–7 m2
(total cross-sectional area) 1 10–2 m (the length of
measuring region in stem)]. The increasing amount of
H215O per 1 cm of the internode was 5.18 0.47 10–2 ml s–1,
which was calculated from the linear part of the slope in
Fig. 2. The amount of H215O continued to increase and
occupied a volume of 40 ml after 15 min, which was420 times
Gamma ray detector
Bi4Ge3O12 crystal
coincidence
circuit
detector
to T.S.C.A
computer
A.D. converter
PMT
linear amp
preamp
timing S.C.A
fast & slow coincidence
rate meter
Fig. 1 A diagram of the measurement system using H215O. H215O
in the vial was applied to the cut shoot. A pair of gamma ray
detectors was adjusted to the position for measurement, 2 cm
above the cotyledon. Sample plants are about 25 cm in height.
In the experiment, a long cable, about 4 m, was used to connect the
gamma ray detector with the coincidence circuit. The vial and
detectors are shielded by lead blocks. The illustrated parts, except
for the coincidence circuit, are set in the growth chamber. S.C.A,
single channel analyzer; PMT, photo multiplier tube.
A
greater than the vessel capacity (Fig. 2A). This observation
indicates that large amounts of water continuously escaped
from the xylem vessels to the surrounding tissues. Lateral
water movement associated with its longitudinal transport
has been reported for some other plant species (Raney
and Vaadia 1965, Trip and Gorham 1968, Bull et al. 1972).
Thus, the same phenomenon was observed in an independent sample in the same manner as H215O. THO spread
throughout the transection of the stem within 10 min.
In contrast to our expectations, a high amount of THO
was not observed in the vascular ring but in the pith.
This indicates a rapid inward movement of water independent of the transpiration. The newly introduced water
containing THO could not have moved by diffusion alone
to 10 cm from the application point within 10 min, given
the average diffusion distance of about 1.7 mm (l2 ¼ 2Dt,
where l is the average diffusion distance, D is the diffusion
coefficient, 2. 4 10–9 (m2 s–1), and t is the measuring time).
Thus, THO must have been translocated mainly through
the xylem vessels followed by diffusional escape at the
point of measurement (Fig. 2B).
To corroborate this conclusion, lateral water movement was examined in detail. Cut internode ends, 1 cm
below the cotyledon, were placed into non-labeled water
after THO was applied for 5 s. Fig. 3 shows the radiograph
of THO by pulse application. A high accumulation
of THO was observed in xylem vessels just 5 s after
60
B
total water volume in the stem internode
volume (µl)
45
30
15
total xylem vessels volume
0
0
200
400
600
time (s)
800
1000
(N=5)
Fig. 2 (A) The volume occupied by H215O water in the internode at the position 2 cm above the cotyledon. The vertical axis indicates the
H215O volume in the stem per cm. H215O was supplied at time point 0. Each symbol represents the data of individual cut shoots (N ¼ 5).
The dotted and solid lines show the total volume of the water and the maximum capacity of the vessels in the sample seedlings per 1 cm,
respectively. The total water volume of that section was about 49–56 ml, based on the diameter of the sample stem (2.9 0.10 mm), and a
water content of 83%, estimated from the dry and fresh weights of the sections. (B) Radiograph of THO at the stem transection. The upper
radiograph shows the THO distribution in the transection of the stem (lower image) at the measuring position after perfusion of THO. THO
was applied to the samples for 10 min in the same manner as H215O. The density of the radiograph is presumed to be proportional to the
amount of radioactivity. Bar ¼ 1 mm. The average of the density of the radiograph of the control, stem without THO, was the same level as
that of no sample, i.e. background level.
Lateral water movement in soybean stems
0s
10 s
20 s
1 min
0
THO (5 s)
721
2 min
1
Fig. 3 Radiographs of stem sections
(upper images) of the THO distribution
in the transection of the stem at the
position of measurement for H215O,
2 cm above the cotyledon, after pulse
application of THO to cut soybean
shoots. The density is presumed to be
proportional to the amount of radioactivity. The cut shoot end, 1 cm below
the cotyledons, was dipped into THO
for 5 s. The samples used in this
experiment were grown in the conditions described in Materials and
Methods. The micrographs (lower
images) represent a transection about
2 mm from the radiography site.
2
H2O
time (min)
stem
(a)
(b)
vessel
(c)
(d)
phloem
Fig. 4 Schematic illustration of the four possible routes for water movement after escape from xylem vessels. Upward pointing arrows
represent the flow of H215O transported through the xylem vessels. The other arrows represent the directions of escaped H215O, i.e. lateral
water movement. The darker shading corresponds to a putatively higher concentration of H215O. (a) Water flows towards the stem surface
and evaporates; (b) water flows into the sieve tubes and is transported downward; (c) water flows through tissue other than xylem vessels;
(d) water re-enters the xylem vessels.
its application. The region with a high accumulatiton of
THO expanded to the whole xylem area after 10 s. After a
further 10 s, a high accumulation of THO had progressed
in a horizontal direction. One minute after the pulse
treatment, the radiograph of THO was similar to that
after 10 s, i.e. the accumulation in a horizontal direction
observed at 20 s had disappeared. Two minutes after the
pulse treatment, the distribution of THO had expanded to
the whole transection. On the radiographic image, the
intensity of the dark image was less at 10 min after
treatment as compared with 0, 10 and 20 s after treatment.
It was therefore concluded that the total amount of THO
in the transection decreased. This shows that xylem vessels
were used for longitudinal water transport. The water
escape must have been ‘flushed’ away quite rapidly since
the radioactivity disappeared with time (Fig. 3). Unfortunately, the route of lateral water movement (apoplastic
or symplastic) could not be identified due the lack of
resolution. Moreover, it was not clear whether the blurring
of the radiographic images was caused by technical or
biological factors, because movement of the THO for 1 min
by self-diffusion alone is about 550 mm.
Next, we analyzed in detail to which tissues the water
was transported after escaping from the xylem vessels.
Below four models describing these possible destinations
for the escaped water are discussed: (a) the water flows
towards the stem surface and evaporates; (b) the water is
collected and transported downward by phloem; (c) the
water flows upward through the entire transection of
the internode except for xylem vessels and phloem; and
(d) the water re-enters the xylem vessels (Fig. 4a–d).
Water in stem tissues easily evaporates through the
surface (Yamamoto 1995). The evaporation from the stem
surface may act as a driving force in the outward radial
water transport. However, the model (a) cannot be valid,
because no change in H215O escape was observed after the
surface of the internode was covered with Vaseline to
prevent evaporation (Fig. 5A).
The second model (Fig. 4b) finds support in the fact
that water is exchanged between xylem and phloem
(Köckenberger et al. 1997, Patrick et al. 2001). In addition,
the high osmolarity of the sieve tube contents may drive
radial flow of water (Van Bel 1990). However, the second
potential route is discounted here because no change in
H215O escape was measured when the tissue outside of the
cambium was removed to knock out phloem transport
(Fig. 5B).
The model (c) (Fig. 4c) was excluded for the following
reasons. The average total cross-sectional areas of xylem
P Pm
vessels ( SD), N1 N
1
i¼1 SVi ðxÞ, at 2 cm (position
722
Lateral water movement in soybean stems
A 60
B 60
stem
45
volume (µl)
volume (µl)
45
30
vessel
15
0
stem
30
phloem
15
0
200
400
600
time (s)
800
1000
0
0
200
400
600
time (s)
800
1000
Fig. 5 (A) The effect of the Vaseline treatment on lateral water movement. Water evaporation from the stem surface was inhibited by a
Vaseline cover on the surface. The vertical axis indicates the volume in the stem occupied by H215O per 1 cm. H215O was applied at t ¼ 0.
The filled circles and open squares quantify the volume of the H215O before and after applying Vaseline, respectively. The experiment was
repeated three times. (B) The effect of the presence of the lateral displacement of water. Phloem flow was knocked out by removing the
tissue outside of the cambium at both sides of the measuring position. The vertical axis indicates the volume in the stem occupied
by H215O per 1 cm. H215O was applied at t ¼ 0. Filled circles and crosses indicate the volume of the H215O before and after removing
the tissue outside of the cambium, respectively. The experiment was repeated three times. Schematic illustrations in the figure represent the
potential scenarios corresponding to the experiments (a detailed explanation was described in the text, referred to in Fig. 4).
of measurement) and 6 cm above the cotyledon were
1.91 0.32 10–7 m2 and 1.86 0.31 10–7 m2, respectively
(where Svi is the area of the ith xylem vessel, m is the total
number of xylem vessels of one sample, N is the total number of samples ¼ 6, and x is the sampling position of the
transection ¼ 2 or 6 cm). To eliminate the variation between
the samples, other indicators were
calculated as follows.
Pm
P N P m
1
The average
of
the
ratios
Vi ð2Þ= i¼1 SVi ð6Þ
1
i¼1 S
N
P
P
P
m
m
2
2
and N1 N
were 1.03 0.08
1
i¼1 ðSVi ð2ÞÞ = i¼1 ðSVi ð6ÞÞ
and 1.07 0.11, respectively (N ¼ 6). The value of SVi2
is proportional to the sum of the biquadrate of the diameter of each xylem vessel; namely, it corresponds to the
capacity of water transport. Both values were almost
equal to 1. Therefore, it is plausible to assume that the
capacity of water transport remains equal between the
three measuring points, although the architecture of xylem
vessels usually changes along the internode. For simplicity,
we regarded xylem vessels as pipes with many surface
pores, and imagined that water was transported through
them. If the water which escaped from the pipe did not
return to it, the amount of water transported should
decrease with height along the pipe. Furthermore, if the
capacity of the pipe to transport water (such as the diameter
of the pipe and the proportion of water escaping) were
constant, the decrease in the amount of water transported
through it, i.e. the flow rate (ml s1), results in a decrease
in the flow velocity (mm s1) through it. Conversely, if the
flow velocity was kept constant in a region, the flow rate
would be constant, too. The flow velocity of water through
xylem vessels could be approximately estimated from the
time delay of the H215O to reach each measuring points.
In addition, it was confirmed that the H215O initially
detected would have passed through xylem vessels, because
a high level of accumulation of THO was observed at the
xylem vessels (Fig. 11).
The profiles of H215O distribution along the internode
(Fig. 6) were similar, although the regression line decreased
with the distance between the measuring point and the site
of application. However, the periods needed to transport
detectable amounts of H215O to the respective measuring
points (intersections between d and e, as well as e and f)
suggest that the flow velocity of H215O through the xylem
vessel was almost constant (4 mm s–1) (Fig. 6).
There is a second reason to discount the third model.
The ratio of H215O escape could be calculated (¼ the
amount of water which escaped from the xylem vessels/the
total amount of water transported through the xylem
vessels). If we regard the total flow rate as approximately
equal to the transpiration rate, the escape rate of H215O
from the xylem vessels was 0.052 ml s–1 (Fig. 2A). The
transpiration rate of the sample under the same conditions
was 0.91 0.13 ml s–1. Therefore, the percentage of water
that escaped from the vessel flow was 5.7 0.82% cm–1.
Provided that this ratio (about 6%) is maintained along
the entire internode length of 8–9 cm, the total amount of
water escaping from xylem vessels is 440% (5.7 8.5 ¼
48%) of the vessel water transported. If the other part of the
internode, 8 cm below the cotyledons, had also been taken
into consideration, the estimate would be 490%
(5.7 16.5 ¼ 94%).
Lateral water movement in soybean stems
stem
1000
800
65 mm
c
b
300
relative count
a
relative count
723
600
400
d
200
e
50 mm
f
35mm
100
0
10
20
30
time (s)
40
50
200
0
0
20
40
60
80
time (s)
In addition, tissues other than xylem vessels are
indirectly involved in mass flow. In tomato plants, the
volume of the AFS (apparent free space), which was
associated with mass transport, was 2.5 times as large as
the volume of the xylem vessels (Van Bel 1978). However,
the mass flow of water through xylem vessels could be
described by the Hagen–Poiseuille law (DeBoer and Volkov
2003); the flow rate is proportional to the fourth power of
the tube diameter. The water path other than through xylem
vessels has comparatively low water conductivity. Therefore, about 40% of the total volume of water transported
through xylem vessels could not possibly be transported
through compartments other than the xylem vessels.
In addition, upward water flow observed by MRI was
only reported to follow the xylem vessels (Kuchenbrod
et al. 1996, Rokitta et al. 1999, Scheenen et al. 2002).
In conclusion, model 3 is ruled out to explain the behavior
of water, although evidence for its rejection is indirect
(Fig. 4c).
In accordance with the rejection of the above three
models, most of the water which escaped from xylem vessels
must re-enter the xylem vessels along the internode (Fig. 4d),
i.e. there is an intense and permanent lateral water
exchange between xylem vessel and the surrounding tissues
along the upward pathway. According to this scenario,
the decrease in the gradient of H215O uptake along the
internode (Fig. 6) has to be interpreted as a decrease in the
H215O concentration in xylem vessels rather than a
decrease in water escape. The interpretation does not
contradict previous reports on water movement analyzed
100
Fig. 6 The flow rate of H215O which escaped
from the xylem vessels at different positions
above the cotyledon. The amount of H215O
which escaped was measured at three positions in the stem, 35, 50 and 65 mm above
the cotyledon (a, b and c, respectively). The
intersection of the curve at the x-axis, after
elimination of the noise, shows the time when
H215O was first detected, as indicated by d,
e and f. From the time difference between d
and e, and between e and f, the flow velocity
of water was calculated to be about 4 mm s–1.
Schematic illustrations in the figure represent
the potential scenarios corresponding to the
experiment (a detailed explanation was
described in the text, referred to in Fig. 4).
with THO (Biddulph et al. 1961, Bull et al. 1972, Van Bel
1974, Van Bel 1976). In the next section, a mathematical
model based on these experimental results, i.e. the fourth
model, is designed to analyze the water exchange in detail.
The model of water exchange in a stem
The results showed that water was mainly transported
through the xylem vessels, and was mixed with water from
the surrounding tissues (Fig. 4d). To construct a mathematical model, we made the following plausible assumptions: (i)
H215O in the xylem vessels was diluted with H216O (innate
water) from the adjacent tissues during upward transport;
(ii) the diameter of the internode at the measuring positions
was constant during the measurement, within 20 min; and
(iii) the 15O was only present as H215O. To examine the
third assumption, the exchange effect of the isotope, 15O,
was studied by mixing it with cellulose powder. The
exchange rate was 0.97 0.021 (N ¼ 5); therefore, the
effect in the plant must be negligible in the experiment
because the effect of the surface of cellulose powder on the
isotope exchange is much greater than that for the plant.
The model includes the following variables: the water
volume exchanged per unit time, V (m3 s–1); the volume of
H215O outside xylem vessels, i.e. newly applied water, A15
(m3); and the volume of H216O outside xylem vessels,
i.e. innate water, A16 (m3).
When the volume of the internode is constant during
the measurement, then
A16 þ A15 ¼ A ðm3 Þ
ð1Þ
Lateral water movement in soybean stems
where A indicates the total volume outside the xylem
vessels (m3).
In the same manner as equation (1), if the volumes of
H215O (m3) and H216O (m3) inside the xylem vessels are set
as B15 and B16, respectively, then
ð2Þ
B15 þ B16 ¼ B m3
where B represents the total volume inside the xylem
vessels (m3).
The exchange rate of A16 for B16 is proportional to the
concentrations of both A16 and B16.
dA16
B16 A16
¼V
Therefore
ð3Þ
dt
B
A
To simplify equation (3), we introduced another
assumption: (iv) the concentration of H215O in the xylem
vessels was kept constant for a while after the innate water
of the xylem vessel was flushed out due to the following
reason. The content of H215O in the re-entered water should
be low, and may be regarded as negligible, at an early stage
after H215O application, because the amount of H215O in
the surrounding tissues was much lower than that of H216O.
Thus, the concentration of H215O in the water transported
to the point of the measurement would be stable while the
condition was maintained. Consequently, we regarded the
concentration of H215O (B15/B) as constant, indicating that
B16/B was also constant. The relationship can be described
by a constant, C (B16/B ¼ C/A ¼ const). Equation (3) is then
as follows:
dA16
V
¼ ðA16 CÞ
A
dt
Equation (4) can be solved analytically, then
A16 ¼ M0 et= þ C
ð4Þ
ð5Þ
where M0 and ¼ A=V indicate the initial value and time
constant, respectively.
The measurable value by the detector, M, is only the
volume of H215O, i.e. A15 þ B15. By substituting equation
(5) in equation (1), and by adding B15, M becomes
M ¼ M1 M0 et=
ð6Þ
where M1 ¼ B15 þ A – C.
The values measured are consistent with the curve
described in equation (6) (Fig. 7), which shows that the
model describes the phenomena adequately.
Calculation of the dilution and exchange rate
If the time course of the volume of the H215O at
the measuring point could be described by equation (6),
a linear relationship could be established when the elapsed
time, t, was small compared with the time constant ,
900 140 s. In fact, a linear increase was observed in the
initial part of the H215O uptake curve (Figs. 2, 5, 6, 9).
80
M = M1 − M0e −t τ
R2 = 0.98–0.99
60
volume [µl]
724
40
20
0
0
500
1000
1500
time [s]
Fig. 7 The fitted curve based on equation (6). Data are derived
from those presented in Fig. 2.
As described above (referred to the text in Fig. 4c), the
capacity of water transport along the internode was
approximately the same; therefore, the escape rate of
H215O from xylem vessels must be the same as well along
the internode.
In the H215O uptake curve, it becomes clear that
when the gradient of the curve, , is proportional to the
concentration of H215O(C), transported through xylem
vessels, the equation
¼ C
ð7Þ
is applicable, in which a represents the proportional
coefficient. In addition, the amount of escaped water
showed linearity in the early stage of water exchange
(Figs. 2, 5, 6, 9), indicating that the concentration of H215O
in xylem vessels could be regarded as constant in this stage.
H215O was diluted due to exchange with the innate
water around the xylem vessels. Thus, the dilution rate per
unit-distance transport, j, could be described as follows,
when H215O was transported from P1 to P2.
1 1d
CðP2 Þ d
2
ð8Þ
¼
’¼
CðP1 Þ
1
in which P1 and P2 represent the position of the detectors,
and d is the distance between P1 and P2, and the flow rates
of H215O escape at the positions are 1 and 2, respectively
(Fig. 8). Therefore, the dilution rate, j could be calculated
from the value of the gradients, at P1 and P2. The
exchange rate Z, which indicates the rate of water escape
per unit-distance of longitudinal transport, is also calculated by
¼1’
ð9Þ
In addition to the H215O, we investigated the uptake of
fluorine (18F), which has often been used as a tracer for
Lateral water movement in soybean stems
apoplastic transport (Mckay et al. 1988, Keutgen et al.
2002), to compare both the uptake profiles. A 1/5 Steinberg
solution containing 18F was applied to the sample in the
same manner about 1 h after of the H215O application. Fig. 9
shows the rates of water and fluorine (18F) escape from the
xylem vessels in the same internode. The profile of the escape
rate of 18F was clearly different from that of water. An early
phase of linear escape was not observed and the curves at
P1 and P2 were almost the same. 18F also must have been
translocated mainly through the xylem vessels followed by
diffusional escape at the point of measurement, like water,
because the diffusional distance for this measurement
period is so short. The fluorine which escaped from xylem
vessels may move via the apoplastic route described above,
internode
P2
35 mm
P1
water volume
flow rate of water escape
P1
∆1
∆2
ϕ = (Cn / C0)1/n
ϕ2C0
VðxÞ ¼ 1:46 106 x2 1:66 105 x
ð10Þ
in which V and x indicate the transpiration rate and the
C0
∆ ∝ C[H215O]
The effect of the transpiration rate on the net volume of water
exchange
To study the effect of the transpiration rate on the net
volume of water exchange, the relationship between the
transpiration rate and relative humidity (RH) was examined
(Fig. 10). The relationship could be described by the second
degree curve based on the equation,
þ 0:0194 ðR2 ¼ 0:9Þ
ϕC0
Pn
∆n
Cn = (ϕ) n C0
because the uptake of fluorine by living cells is very slight,
in the same manner as inulin (Mckay et al. 1988). The uptake
profile of 18F was shown to be independent of the position
of measurement, indicating that the dilution effect of 18F
during its transport through the xylem vessels was slight,
unlike water. In addition, the dilution of water due to
exchange was not negligible; the dilution rate and the rate
of water exchange of this sample were 85.4 and 14.6%,
respectively, in this case (Fig. 9a).
flow
ϕ = (∆2/∆1)1/3.5
(/ cm)
P2
ϕnC0
725
time
Fig. 8 Calculation of the dilution rate of H215O along the
internode. The flow rate of the water escape is represented by n
(in the linearity region) as measured by the detector setting at
position Pn. C is the concentration of H215O transported through
the xylem vessels. j is the dilution rate of water during its transport
upward which is obtained simply by measuring the gradients, at
P1 and P2, and calculating their ratio, 2/1. The distance between
P1 and P2 was 35 mm in this experiment.
(a) 4 × 104
RH, respectively. To compare the net volumes of water
exchange in plants under different humidity conditions, one
should pay attention to the fact that the flow rate through
xylem vessels is strongly affected by the RH conditions at
the time of measurement. Therefore, the transpiration rate
of plants under a certain RH regime could be estimated
by equation (10). The net volume of water exchange, (x),
under a certain RH, x, could be estimated by the rate of the
exchange, Z(x), and the transpiration rate, V(x), as follows:
ðxÞ ¼ ZðxÞ VðxÞ
ð11Þ
(b) 2000
P2
3 × 104
P1
35 mm
1500
relative
P1
2 × 104
1000
P2
1 × 104
0
500
0
200
400
600
time (s)
800
1000
1200
0
0
200
400
600
800
1000
1200
time (s)
Fig. 9 The influence of the measuring position on the measured flow rate of water and the amount of fluorine. (a) Quantification of water
escape at position P1 (open squares) and P2 (filled triangles) in the plant and (b) the escape of 18F at the same positions in the plant after the
application of H215O.
726
Lateral water movement in soybean stems
The rate of (x), r,
r ¼ ðx1 Þ=ðx2 Þ
ð12Þ
was calculated from the measurement of water exchange
under various RH conditions (Table 1). The value of r
was more or less constant, 1.05 0.21 (N ¼ 11, SD).
In conclusion, the net volume of water exchanged with
that in the surrounding tissue was independent of the
transpiration rate.
Van Bel (1974, 1976) claimed that water escape can be
explained by the Horwitz model based on the diffusion
theory (Horwitz 1958). THO was allowed to perfuse
through stem segments of tomato plants under gravity,
and the rates of THO re-collected in the outflow and THO
remaining in the respective stem sections were measured
(Van Bel 1974). He concluded that the higher the apparent
flow velocity, the larger the amount of the THO molecules
not available for lateral diffusional escape (Biddulph et al.
1963, Van Bel 1974), because the rate of re-collected
THO appeared to be highly dependent on the flow rate
of the perfusion. Some of his graphs suggested a reversibility of the escape, i.e. re-entrance of THO into the
xylem vessels. In the present experiments, the net volume of
exchanged water was almost constant in spite of the changes
in the mass flow rate. The interpretation is in agreement
with previous reports that the outflow constant in the
Horwitz theory was close to the water diffusion coefficient
(Van Bel 1974).
To establish diffusional escape as the driving force for
lateral water movement, samples were immersed in water at
0 or 278C for 5 and 10 min after the pulse application of
THO. In this treatment, the whole shoot was frozen before
excising the internode segment to avoid the drastic change
in the pressure in xylem vessels due to cutting near the
target point. Fig. 11 shows the radiograph of THO at the
transection of the stem of the samples. A large amount of
THO was observed in xylem vessels after 5 s (Fig.11a). THO
could reach the whole area of the transection within 5 min
0.025
5 min
10 min
transpiration rate (water g/cm2/h)
(b)
(a)
0.02
27°C
(c)
0.015
0°C
0s
THO (5 s)
0.005
20
40
60
relative humidity (%)
80
100
Fig. 10 The relationship between the rate of transpiration of
plants and the relative humidity. The rate of transpiration, V, can be
described by the second degree equation of relative humidity, x,
V(x) ¼ –1.4610–6 x2 – 1.6610–5 x þ 0.0194 (R2 ¼ 0.9).
Table 1
10
5
0.01
time (min)
in water
Fig. 11 Radiographs of the THO at the stem transection. The
radiographs show the THO distribution in the transection of the
stem near the measuring position. The darker regions in the images
mark higher amounts of radioactivity. THO was applied to the cut
end of shoots, 1 cm below the cotyledons, for 5 s (a). The respective
shoots were then immersed in water at 278C (room temperature) (b)
or 08C (c) for 5 and 10 min, respectively, and were frozen and
exposed to the imaging plate. Bar ¼ 1 mm.
The net volume of water exchange under various relative humidities in cut soybean shoots
Sample number
RHL
j
RHH
j
r
1
2
3
4
5
6
7
8
9
10
11
52.0
0.939
78.0
0.892
0.895
29.5
0.852
61.0
0.810
1.06
35.8
0.973
51.5
0.977
1.35
55.0
0.953
77.1
0.928
0.972
53.0
0.965
82.7
0.944
1.05
51.8
0.930
82.7
0.915
1.48
56.1
0.847
74.5
0.807
0.918
54.0
0.910
80.0
0.840
0.917
33.5
0.948
75.6
0.880
0.756
24.0
0.960
50.6
0.950
0.975
31.3
0.865
48.2
0.869
1.19
RH, relative humidity; j, dilution rate; r, rate of (x), (xlow)/ (xhigh); (x), net volume of water exchange; x, relative humidity. The rate,
r, can be estimated by measuring j for the same sample under different relative humidities.
Lateral water movement in soybean stems
under both conditions (0 and 278C) after the THO pulse
treatment (Fig. 11b, c). The profile of the radiographs of
THO under 278C conditions was similar to the previous
results (Fig. 2B), i.e. local accumulation of THO at pith
was detected, supporting the proposal that rapid inward
movement of water was independent of the transpiration.
On the other hand, the profile under 08C conditions
showed uniform THO distribution (Fig. 11c). In addition
to the self-diffusion (thermal motion), some driving
force may be involved in the lateral water movement in
this case, because an average distance of water movement
by self-diffusion for 5 min at 08C is 0.8 mm (l2 ¼ 2Dt,
D ¼ 1.1 10–9, 2.4 10–9 (m2 s–1) referred to in Fig. 2), i.e.
the distance is too short to explain the profile of the
radiograph (Fig. 11c). The average distances of water
movement by self-diffusion for 10 min at 0 and 278C are
1.1 and 1.7 mm, respectively, suggesting that the period
is enough for THO to reach the whole area in an internode transection. However, the radiograph at 278C was
clearly different from that at 08C. The water movement
within a stem may be strongly affected by plasma
membrane partitioning, because the water permeability
of the plasma membrane is dependent on the temperature
(Lee et al. 2005).
Materials and Methods
Sample preparation
Soybean seedlings [Glycine max (L.) Merr. cv. Enrei] were
germinated in vermiculite at 278C in the dark. After 3 d, the
seedlings were transferred to a 1/5 Steingerg solution and were
grown in 80% RH at 278C, with a 16 h light (150 mmol m–2 s–1) and
8 h dark cycle (Biotron LPH300; NK System Co. Osaka, Japan).
The culture solution was renewed every week. Samples aged from
18 to 20 d old were used for this experiment.
Measurement of the vessel area
To measure the vessel area, a 0.05% safranine solution was
applied to the plant for 30 min with 50% humidity at 288C, with
light. Stem sections of 150 mm thickness were cut with a vibratome
(VT1000S; Leica Microsystems Co., Tokyo, Japan) at 2 and 6 cm
above the cotyledon. The section was photographed under a
microscope and the image transferred to a computer, by which the
xylem vessel area was measured using an image-analyzing software
program (IPlab; Scanalytics, Inc., Fairfax, VA, USA).
Generation of H215O
The gas 15O2 was generated by the reaction of 14N (d, n)15O;
the H215O vapor was then synthesized by the reaction of the
oxygen (15O) and hydrogen gases under high temperature in the
presence of a platinum catalyst. The H215O was captured by
bubbling the vapor through a bottle filled with 10 ml of distilled
water. The H215O was used in the present experiment after the
radioactivity of the H215O was measured using a dose calibrator
(Atomlab 100A; Biodex Medical Systems Co., Shirley, NY, USA).
The specific activity of H215O was 2 GBq per 10 g of water.
727
The quantitative and real-time measurement of H215O in the stem
internode
A soybean plant, from which the cotyledons and the root
8 cm below the cotyledons had been removed, was fixed between
lead blocks to shield radiation, and placed in the Biotron about 1 h
before the measurement. A pair of gamma ray detectors was placed
immediately adjacent to either side of the internode of the plant,
2 cm above the cotyledons. The detector modules (Photosensor
Modules; Hamamatsu Photonics K. K., Hamamatsu, Japan)
were composed of a BGO scintillator (Bi4Ge3O12 crystal size
10 10 20 mm), photomultiplier tube and pre-amplifier. The
gamma rays radiated from the sample were converted to the weak
current by the modules. The weak current was amplified by a linear
amplifier (704-4B; Oken Co., Tokyo, Japan), and discriminated by
a timing single channel analyzer (706-2B, Oken Co.) based on its
energy, corresponding to 511 keV. The signals passed through the
timing single channel analyzer were counted with a coincidence
circuit (Fast and slow coincidence 708-1B and Ratemater S-2293B;
Oken Co.) and recorded on a computer (OptiPlex GX1; Dell Co.
Kawasaki, Japan) (Fig. 1). The timing of coincidence and the data
export interval were set at 110 ns and 1 s, respectively. The counting
efficiency of the gamma ray detector was calculated to be 0.120%,
by comparing the count of the gamma ray detector with that of
a gamma counter (Auto-gamma scintillation spectrometer 5230;
GMI Inc. Ramsey, MN, USA) for the plant sample supplied with
H215O (200 MBq ml–1). The measuring system was placed in a
Biotron, which was maintained at 278C with an RH of 50% and
constant light. The background noise was reduced to 50.01 c.p.s.
by the coincidence circuit and the shielding of the detection unit
with lead blocks.
The measurement of H215O volume was performed on plants
with two treatments: (i) a plant with Vaseline spread on the surface
of the internode to be measured between the cotyledon and the
first leaf; and (ii) a plant with a 5 mm portion of the tissue
outside of the cambium removed by a razor blade, from 3 cm above
to 1 cm below the measuring position. After the measurement,
the sample of tissue removed from outside of the cambium was
treated with 0.05% safranine solution for 30 min. Both sections of
the removed tissue, 150 mm in thickness, were observed under the
microscope to verify the condition of the remaining phloem and
the associated vessels.
Quantitative measurement of water volume by two pairs of gamma
ray detectors
Two pairs of gamma ray detectors were placed close to either
side of the internode of a soybean plant, 2 cm above the cotyledon
(P1) and 3.5 cm above P1 (P2), respectively. The H215O was applied
and measurements were carried out under a range of RHs from
24 to 83%, as described above.
A supplementary experiment was executed using a doublelabel approach: a 1/5 Steinberg solution containing 18F was applied
to the same sample about 1 h after the 1/5 Steinberg solution
containing H215O was applied.
Radiography of THO distribution over the transection of the
internode
To examine the distribution of water within an internode
transection, THO (Moravek Biochemicals Inc., Brea, CA, USA),
3.2 MBq g–1, was supplied in the same manner as H215O. After
10 min, an approximately 1.5 cm long internode segment was
excised below the measurement site, 2 cm above the cotyledons,
and immediately frozen by immersion in liquid nitrogen.
728
Lateral water movement in soybean stems
Following embedding in Tissue-Tek at –228C, the sample was
shaved to flatten the transection using a cryostat (Microm
HM505N; Carl Zeiss Co., Tokyo, Japan) and exposed to an
imaging plate (BAS-TR2025; Fujifilm CO., Tokyo, Japan) for
2 weeks at 808C. The image from the imaging plate was scanned
with an image analyzer (FLA-5000, Fujifilm Co. Japan) with a
16-bit data depth.
To examine lateral water movement in the internode, the cut
shoots were placed into non-labeled water for various times (10 s,
20 s, 1 min and 2 min) after the cut ends were dipped for 5 s into a
THO solution. Then an approximately 1.5 cm internode segment
was excised above the measurement site and frozen by immersion
in liquid nitrogen. The following procedures were as described
above. THO (37 MBq g–1) was applied at the cut end of the stem,
1 cm below the cotyledon. The exposure time to an imaging plate
was 1 d at –808C.
To examine diffusional lateral water movement in the
internode, THO (37 MBq g–1) was applied to the cut end of the
stem, 1 cm below the cotyledon, for 5 s. Three treatments were then
carried out: (i) the sample was immediately frozen then exposed to
an imaging plate; (ii) the sample was immersed in water at 08C for
5 or 10 min followed by freezing and exposure to an imaging plate;
and (iii) the sample was immersed in water at 278C (room
temperature) for 5 or 10 min followed by freezing and exposure to
an imaging plate.
Measurement of the transpiration rate
Cotyledons and roots, 8 cm below the cotyledons, of soybean
plants were removed. The cut shoot was placed in a plastic bottle
containing 300 ml of distilled water. The bottle was set in the
Biotron, which was kept in the growth conditions described above.
Transpiration rates were measured by monitoring weight loss. The
leaves of the cut shoot were removed and their surface area was
measured using a scanner (N676-U; Canon Inc. Tokyo, Japan).
The measurement of the effect of the exchange of the isotope 15O
for 16O
To measure the effect of the exchange of the isotope 15O for
16
O, 8 ml of H215O was poured into a vial filled with 2 g of cellulose
powder (Fibrous cellulose powder CF11; Whatman Japan K.K.,
Tokyo, Japan). They were mixed homogeneously by a shaker
(Vortex genius3; IKA Japan K.K., Yamatokoriyama, Japan) for
about 5 min. Then, 50 ml of supernatant water was sampled after
centrifuging for 1 min. The activity of the water was measured
by a gamma counter (Auto-gamma scintillation spectrometer
5230; Packard Co.), and the ratio (the activity of the supernatant
water/the activity of the initial H215O water) was calculated.
The experiment was repeated four times.
Funding
The National Food Research Institute.
Acknowledgments
We wish to thank Professor Iiyama for his valuable
suggestions, and Makoto Takei, Nobuki Nengaki and Masanao
Ogawa for valuable technical assistance, especially for supplying
H215O at the National Institute of Radiological Sciences. This
study was a part of the Development of Nanotechnology and
Materials for Innovative Utilizations of Biological Functions and
supported by the Ministry of Education, Culture, Sports, Science
and Technology, Grants-in-Aid for Scientific Research (A).
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(Received January 28, 2008; Accepted March 18, 2008)